Stability of enzyme immobilized on the nanofluidic channel surface

The lifetime of an enzyme is critical to prevent system failure and optimize maintenance schedules in biological and analytical chemistry. The lifetime metrics of an enzyme can be evaluated from enzyme activity in terms of catalytic cycles per enzyme at various storage times. Trypsin, which is a gold-standard enzyme in proteomics, has been known to decrease activity due to self-digestion. To improve the activity of trypsin, enzyme reactors have developed by immobilizing in micro and nanospace. However, an evaluation method for the catalytic cycle has not been established due to major issues such as nonuniform space, unstable liquid transport, and self-digestion during immobilization in conventional work. To solve these issues, we have previously developed an ultra-fast enzyme reactor with a well-defined nanofabrication method, stable liquid transport, and partial enzyme modification. Here, we aimed to investigate catalytic cycles in a nanochannel. To extend enzyme lifetime efficiently, we have evaluated the optimal immobilization process and catalytic cycles of trypsin. As a result, immobilized enzyme densities by the trypsinogen immobilization process were increased at all concentrations compared to the trypsin immobilization process. To evaluate the lifetime of trypsin, the immobilized enzyme densities and activities were almost the same before and after 72 h of enzyme storage, and the calculated catalytic cycles were 1740. These results indicated that self-digestion of the immobilized enzyme was highly suppressed. Consequently, the reaction efficiency has been evaluated depending on the catalytic cycles from the substrate for the first time, while preventing self-digestion by trypsin.